Intracutaneous Transplantation Research Articles

Intracutaneous Transplantation of Islets Within a Biodegradable Temporizing Matrix as an Alternative Site for Islet Transplantation.

Human and porcine pancreatic islets were transplanted into a fully vascularized biodegradable temporizing matrix (Novosorb) that creates a unique intracutaneous site outside of the liver in a large-animal preclinical model. The intracutaneous prevascularized site supported pancreatic islet survival for 3 months in a syngeneic porcine-transplant model. Pancreatic (human and porcine) islet survival and function were demonstrated in an intracutaneous site outside of the liver for the first time in a large-animal preclinical model.

Regenerating Hair in Prevascularized Tissue Space Formed by a Controllable Foreign Body Reaction

AbstractIntracutaneous transplantation of trichogenic cells is a currently endorsed strategy to realize hair regeneration in vivo. However, skin is not the most advantageous transplant site due to the robust mechanical property and deficient physiological perfusion. Herein, a subcutaneous space made of prevascularized collagen fibers (PVCF) generated by controlling the duration of in situ foreign body reaction is reported. In contrast to skin, an optimally preprogrammed PVCF presents a larger tissue volume (171 mm3), low Young's modulus (1/2‐fold), high flexibility high mechanical energy dissipation rate (1.3‐fold), highly permeable surface structure, and plentiful vascular network. Remarkably, cells transplanted into PVCF suffer less apoptosis and necrosis, and generate more mature hairs (≈289 per site) than that in intracutaneous transplantation (≈177 per site). The RNA‐sequencing profiling indicates similar trends under molecular level. This work provides a promising strategy to improve graft site microenvironment physiologically and mechanically, with a low‐cost and simple fabrication process.

Effects of platelet-rich plasma on in vitro hair follicle germ preparation for hair regenerative medicine

Hair regenerative medicine is a promising approach for the treatment of hair loss and involves the transplantation of follicular stem cells into bald spots to regenerate hair. Various approaches have been investigated to engineer tissue grafts for use in hair regenerative medicine. Tissue-like three-dimensional aggregates, such as bioengineered hair follicle germs (HFGs), have shown great promise for hair regeneration, with normal tissue morphology and hair cycles. However, these approaches have not yet been applied in clinical settings, and further studies are needed to improve hair generation efficiency. The biological molecules in invivo microenvironments around HFGs may provide cues for the invitro preparation of HFGs with higher trichogenic functionalities. Activated platelet-rich plasma releasate (PRPr) is an autologous source of signaling molecules including growth factors and cytokines. In this study, we investigated the effects of PRPr on the preparation of HFGs invitro. The presence of PRPr did not hinder the spontaneous formation of dumbbell-like HFGs from a suspension of embryonic skin-derived epithelial and mesenchymal cells in a custom-designed HFG culture plate. HFGs prepared with PRPr displayed greater levels of follicular gene expression compared to those prepared in the absence of PRPr. Moreover, the hair regeneration ability upon intracutaneous transplantation was significantly improved in the presence of PRPr. These results suggest that PRPr is beneficial for engineering HFGs for autologous hair regenerative medicine.

The role of regulatory T lymphocytes in immune control of MC-2 fibrosarcoma.

SUMMARYThe role of T regulatory lymphocytes (Treg) particularly in cancer is well known. The goal of the present study was to determine the contribution of these lymphocytes in the regulation of anti-tumor immunity of CBA/HZgr mice against MC-2 fibrosarcoma (4th generation of methylcholanthrene induced tumor). The levels of T lymphocytes (CD4+, CD8+ and CD4+CD25+) were determined 8 and 20 days after tumor transplantation. Further, the role of CD4+CD25+ (Tregs) in tumor-host interaction was evaluated in vitro and in vivo by using specific monoclonal antibodies. We found that splenocytes of both control and Treg depleted tumor bearing mice strongly but differently inhibited growth of tumor cells in vitro. While splenocytes of untreated mice exhibited significant decrease of this activity (from 74.4% to 62.6% and 32.95%), the splenocytes of Treg depleted mice showed increase of this activity (from 79.5% to 84.3% and 86.2%) from day 6 to day 13 and day 21 after tumor grafting, respectively. Further, upon i.v. injecting specific monoclonal anti-Treg antibody tumor immediately prior to tumor cell intracutaneous transplantation, the tumor was rejected after initial growth. In treated mice, the incidence of Treg cells was very low initially, reaching normal values two weeks later. These animals were shown to be resistant to tumor transplantation four months later.

Preparation of hair beads and hair follicle germs for regenerative medicine

Hair regenerative medicine is a promising approach for hair loss, during which autologous follicular stem cells are transplanted into regions of hair loss to regenerate hairs. Because cells transplanted as a single cell suspension scarcely generate hairs, the engineering of three-dimensional (3D) tissues before transplantation has been explored to improve this process. Here, we propose an approach to fabricate collagen-enriched cell aggregates, named hair beads (HBs), through the spontaneous constriction of cell-encapsulated collagen drops. Mouse embryonic mesenchymal cells or human dermal papilla cells were encapsulated in 2-μl collagen microgels, which were concentrated >10-fold in volume during 3 days of culture. Interestingly, HB constriction was attributed to attraction forces driven by myosin II and involved the upregulation of follicular genes. Single HBs with epithelial cells seeded in U-shaped microwells formed dumbbell-like structures comprising respective aggregates (named bead-based hair follicle germs, bbHFGs), during 3 days of culture. bbHFGs efficiently generated hair follicles upon intracutaneous transplantation into the backs of nude mice. Using an automated spotter, this approach was scalable to prepare a large number of bbHFGs, which is important for clinical applications. Therefore, this could represent a robust and practical approach for the preparation of germ-like tissues for hair regenerative medicine.

Spontaneous hair follicle germ (HFG) formation in vitro, enabling the large-scale production of HFGs for regenerative medicine

Hair follicle morphogenesis is triggered by reciprocal interactions between hair follicle germ (HFG) epithelial and mesenchymal layers. Here, we developed a method for large-scale preparation of HFGs in vitro via self-organization of cells. We mixed mouse epidermal and mouse/human mesenchymal cells in suspension and seeded them in microwells of a custom-designed array plate. Over a 3-day culture period, cells initially formed a randomly distributed single cell aggregate and then spatially separated from each other, exhibiting typical HFG morphological features. These self-sorted hair follicle germs (ssHFGs) were shown to be capable of efficient hair-follicle and shaft generation upon intracutaneous transplantation into the backs of nude mice. This finding facilitated the large-scale preparation of approximately 5000 ssHFGs in a microwell-array chip made of oxygen-permeable silicone. We demonstrated that the integrity of the oxygen supply through the bottom of the silicone chip was crucial to enabling both ssHFG formation and subsequent hair shaft generation. Finally, spatially aligned ssHFGs on the chip were encapsulated into a hydrogel and simultaneously transplanted into the back skin of nude mice to preserve their intervening spaces, resulting in spatially aligned hair follicle generation. This simple ssHFG preparation approach is a promising strategy for improving current hair-regenerative medicine techniques.

Hair Follicle Regeneration by Transplantation of a Bioengineered Hair Follicle Germ.

Hair follicle morphogenesis is first induced by epithelial-mesenchymal interactions in the developing embryo. In the hair follicle, various stem-cell populations are maintained in specialized niches to promote repetitive hair follicle-morphogenesis, which is observed in the variable lower region of the hair follicle as a postnatal hair cycle. In contrast, the genesis of most organs is induced only once during embryogenesis. We developed a novel bioengineering technique, the Organ Germ Method, that employs three-dimensional stem cell culture for regenerating various organs and reproducing embryonic organogenesis. In this chapter, we describe a protocol for hair follicle germ reconstitution using adult follicle-derived epithelial stem cells and dermal papilla cells with intracutaneous transplantation of the bioengineered hair-follicle organ germ. This protocol can be useful not only for the clinical study of hair regeneration but also for studies of stem cell biology and organogenesis.

Hair organ regeneration via the bioengineered hair follicular unit transplantation

Organ regenerative therapy aims to reproduce fully functional organs to replace organs that have been lost or damaged as a result of disease, injury, or aging. For the fully functional regeneration of ectodermal organs, a concept has been proposed in which a bioengineered organ is developed by reproducing the embryonic processes of organogenesis. Here, we show that a bioengineered hair follicle germ, which was reconstituted with embryonic skin-derived epithelial and mesenchymal cells and ectopically transplanted, was able to develop histologically correct hair follicles. The bioengineered hair follicles properly connected to the host skin epithelium by intracutaneous transplantation and reproduced the stem cell niche and hair cycles. The bioengineered hair follicles also autonomously connected with nerves and the arrector pili muscle at the permanent region and exhibited piloerection ability. Our findings indicate that the bioengineered hair follicles could restore physiological hair functions and could be applicable to surgical treatments for alopecia.

Fully functional hair follicle regeneration through the rearrangement of stem cells and their niches

Organ replacement regenerative therapy is purported to enable the replacement of organs damaged by disease, injury or aging in the foreseeable future. Here we demonstrate fully functional hair organ regeneration via the intracutaneous transplantation of a bioengineered pelage and vibrissa follicle germ. The pelage and vibrissae are reconstituted with embryonic skin-derived cells and adult vibrissa stem cell region-derived cells, respectively. The bioengineered hair follicle develops the correct structures and forms proper connections with surrounding host tissues such as the epidermis, arrector pili muscle and nerve fibres. The bioengineered follicles also show restored hair cycles and piloerection through the rearrangement of follicular stem cells and their niches. This study thus reveals the potential applications of adult tissue-derived follicular stem cells as a bioengineered organ replacement therapy.

ASKing Macrophages to Promote Hair Growth

Noting that the abundance of ASK1 (apoptosis signal-regulating kinase 1, which is activated in response to inflammatory cytokines and various stresses) increases after epithelial injury in rats, Osaka et al . investigated its involvement in the increase in regional hair growth stimulated by skin injury in mice. Wounds in mice lacking ASK1 ( ASK1 –/– mice) showed apparently normal re-epithelialization, but there was a marked delay in injury-induced hair regrowth. In contrast, hair follicle development in embryos and regrowth after plucking appeared normal. ASK1 is a mitogen-activated protein kinase kinase kinase (MAPKKK), and activation of its downstream targets c-Jun N-terminal kinase and p38 was reduced in ASK1 –/– compared with wild-type mice, as was activation of extracellular signal-regulated kinase (the latter likely through an indirect route). A combination of microarray analysis and reverse transcription polymerase chain reaction (RT-PCR) indicated that the expression of macrophage-specific markers, as well as that of macrophage-specific chemotactic and activating factors, was reduced in the wounded skin of ASK1 –/– animals compared with that in wild-type mice. Immunohistochemical analysis confirmed that ASK1 –/– mice exhibited decreased recruitment of macrophages to the wounded area. Intracutaneous transplantation of bone marrow-derived macrophages (BMDMs) from either wild-type or ASK1 –/– mice stimulated notable hair growth only in wild-type mice; intracutaneous transplantation of cytokine-activated BMDMs, however, stimulated marked hair growth in both wild-type and ASK1 –/– mice. Thus, ASK1-dependent recruitment and activation of macrophages appears to play a critical role in promoting hair growth after injury. N. Osaka, T. Takahashi, S. Murakami, A. Matsuzawa, T. Noguchi, T. Fujiwara, H. Aburatani, K. Moriyama, K. Takeda, H. Ichijo, ASK1-dependent recruitment and activation of macrophages induce hair growth in skin wounds. J. Cell Biol. 176 , 903-909 (2007). [Abstract] [Full Text]

ASK1-dependent recruitment and activation of macrophages induce hair growth in skin wounds

Apoptosis signal-regulating kinase 1 (ASK1) is a member of the mitogen-activated protein 3-kinase family that activates both c-Jun NH2-terminal kinase and p38 pathways in response to inflammatory cytokines and physicochemical stress. We report that ASK1 deficiency in mice results in dramatic retardation of wounding-induced hair regrowth in skin. Oligonucleotide microarray analysis revealed that expression of several chemotactic and activating factors for macrophages, as well as several macrophage-specific marker genes, was reduced in the skin wound area of ASK1-deficient mice. Intracutaneous transplantation of cytokine-activated bone marrow-derived macrophages strongly induced hair growth in both wild-type and ASK1-deficient mice. These findings indicate that ASK1 is required for wounding-induced infiltration and activation of macrophages, which play central roles in inflammation-dependent hair regrowth in skin.

Mixture of trypsin, chymotrypsin and papain reduces formation of metastases and extends survival time of C57Bl6 mice with syngeneic melanoma B16.

The aim of the present study was to investigate the effect of a mixture of proteolytic enzymes (comprising trypsin, chymotrypsin and papain) on the metastatic model of syngeneic melanoma B16. 140 C57B16 mice were divided into two control and two "treated" groups. Control groups received saline rectally, twice a day starting 24 h after intracutaneous transplantation (C1) or from the time point of the primary B16 melanoma extirpation (C2), respectively. "Treated" groups were rectally administered a mixture of 0.2 mg trypsin, 0.5 mg papain, and 0.2 mg chymotrypsin twice daily starting 24 h after transplantation (E1) or after extirpation of the tumor (E2), respectively. Survival of mice and B16 melanoma generalization were observed for a period of 100 days. Immunological evaluation of B16 melanoma cells in the ascites was accomplished. CD44, CD54 and CD106 cells were measured by flow cytometry. Administration of proteolytic enzymes to mice inhibited the growth of primary tumors, and tumor recurrences were less numerous. Importantly, metastasis was considerably curtailed both in the vicinity of the primary tumor and at distant locales. These findings correlated with a decreased expression of CD44 and CD54 molecules in tumors exposed to proteolytic enzymes in vivo. Our data suggest that serine and cysteine proteinases suppress B16 melanoma, and restrict its metastatic dissemination in C57B16 mice.

Regression of Primary Brown-Pearce Testicular Carcinoma and Metastases following Intracutaneous Transplantation with Homologous Tumor

The Brown-Pearce carcinoma is a readily transplantable, highly malignant carcinoma of the testicle of rabbits, first described by Brown and Pearce in 1923 (1). The tumor, when transplanted into the testicle, grows rapidly and produces a generalized carcinomatosis, which usually results in the death of the animal in from three to six weeks. In only rare instances do these testicular transplants and their metastases undergo spontaneous regression. It has been established on various occasions that the Brown-Pearce carcinoma when transplanted into the skin grows but soon regresses, becomes completely resorbed, and leaves the animal refractory to subsequent transplantation with this tumor (2–6). Lumsden (7) has shown that when regression of cutaneous tumor occurs there is a rise in the titer of agglutinins in the serum of the host. The refractory state which develops following intracutaneous transplantation has been confirmed on numerous occasions in this laboratory. In the course of other experiments conducted in this laboratory with the Brown-Pearce carcinoma, it was observed that a distinct regression and even disappearance of previously transplanted and growing testicular tumors and their metastases occurred in animals which had also received transplantations into the skin. The regression of the testicular tumors and their metastases occurred after the inoculated skin tumors had begun to regress. Because regression of the testicular tumors was so striking and because subsequent autopsies showed no evidence of tumor in the testes or at any other site in the body, it was thought advisable to enlarge upon these experiments.